Development of a PCR fluorescence sensor utilizing an upconversion FRET system for rapid and ultra-sensitive determination of Escherichia coli

Abstract

An upconversion-gold nanoparticle detection system that integrates PCR amplification and fluorescence resonance energy transfer was constructed to enable swift and highly sensitive identification of Escherichia coli. The forward primer used in the PCR amplification is modified with sulfhydryl groups, enabling its connection to gold nanoparticles via Au–S bonds. The complementary strand of the forward primer, which is attached to the upconversion nanomaterials, can hybridize with the free forward primer through base complementary pairing. This interaction induces fluorescence resonance energy transfer, resulting in fluorescence quenching. The concentration of the target bacteria influences the amount of free primer in the system after PCR amplification, which subsequently alters the intensity of the upconversion fluorescence. The fluorescent PCR sensor developed based on the aforementioned principles demonstrated a detection limit of 14 CFU/mL for E. coli, with a quantitative detection range of 18–1.8 × 10⁷ CFU/mL. In comparison to the qPCR method, the number of PCR cycles required for the constructed biosensor can be reduced to approximately 22 to achieve the same detection limit, effectively decreasing the detection time by about 24 min. The spiked recoveries in chicken were 91.8–106.0% with the relative standard deviations less than 10%, indicating that the constructed method exhibits good applicability.

Graphical abstract